SUMMARY Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

of continental drift. A relative motion of the continents must involve the mantle to depths of several hundred kilometers; it is no longer possible to imagine thin continental blocks sailing over a fluid mantle.

Many enzymes have been reported to exist in more than one form within the same species. By several methods a number of molecular forms of lactic dehydrogenase (LDH) have been found in differing amounts in the various organs of one individual (1-4). This article describes studies which were undertaken in an attempt to elucidate the molecular nature of these multiple enzymatic forms and to follow their development. Most of the developmental studies were made on the chicken.Two "pure" lactic dehydrogenases occur in the chicken. One of them (CM) is found principally in the breast muscle, and the other (CH) in the heart, of adult chickens. These two enzymes are separate entities as judged by physical, enzymatic, and immunochemical criteria (1, 2, 5). During embryonic development the LDH enzymes of the chicken breast muscle shift from the enzymes related to CH, through several intermediate enzyme types, and appear in the adult as pure CM. The characterization of these intermediate enzyme types by several independent methods has led to the development of the hypothesis, which we present in this article, that the intermediate enzyme types which appear during embryonic development are "hybrid" enzymes, consisting of both CM and CH components. Furthermore, these "hybrids" also occur in the adult tissues of the chicken and in other animals. Immunological ApproachIn order to use immunological methods for the study of the development and structure of specific proteins, it is necessary that the immune systems be 962 fully characterized. Two immune systems were used in this study: (i) CH- (6), immunoelectrophoresis (7), and quantitative precipitation analyses (8, 9). Anti-CH was shown to be heterogeneous when measured by double diffusion in agar. After absorption with CM, two bands remained when tested with crude chicken heart extracts, only one of which showed LDH activity (10, 11). This absorption did not decrease the complement (C') fixation titer of CH-anti-CH (Fig. 1), nor did it remove antibody capable of neutralizing CH enzymatic activity. Thus, the heterogeneity in anti-CH reflected antibodies to impurities in the CH immunizing antigen (probably CM) and not anti-CH cross-reacting with CM. Figure 1 shows the C' fixation curves of CM and CH with anti-CM and anti-CH. After absorption with CM, anti-CH showed no reduction in titer against CH. Moreover, the C' fixation curves were identical when either crystalline CH or crude heart extracts were used as antigen. At the dilutions of antisera used (1/5000 for anti-CH and 1/2200 for anti-CM) no cross reactions between CM and CH are detectable. Figure 2 demonstrates the ability of C' fixation to resolve a mixture of pure CM and CH. The heights of the curves at peak fixation are the same in the mixture as in the two pure systems. By the shift in the abscissa at peak fixation it is possible to calculate the percentage of total enzyme activity which reacts with either anti-CM or anti-CH (9, 12). In an artificial mixture of pure CM and CH (Fig. 2), the percentages calculated by C' fi...

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

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